Dr. Jack Dekkers - Genetics of Host Resistance to PRRS and PCV2
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Genetics of Host Resistance to PRRS and PCV2 - Dr. Jack Dekkers, Iowa State University, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA. More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium
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Dr. Jack Dekkers - Genetics of Host Resistance to PRRS and PCV2
- 1. Genetics of Host Resistance to PRRS and PCV2 Jack Dekkers Department of Animal Science Iowa State University
- 2. Porcine Reproductive and Respiratory Syndrome - PRRS Sows Abortions Stillborn/weak pigs Delayed estrus Respiratory problems Grower Increased mortality Decreased production Respiratory problems Large financial loss in both production settings Respig.com Respig.com Eradication Biosecurity Vaccination Host genetics Strategies to control PRRS
- 3. Objective Use genomics to identify genes / genomic regions associated with resistance / susceptibility to PRRS virus infection Led by Joan Lunney – USDA – ARS Beltsville Bob Rowland – Kansas State University Jim Reecy & Jack Dekkers – Iowa State University Strong Industry Participation PHGC Breeding Companies Fast Genetics, Genesus, Choice Genetics PIC/Genus, TOPIGS, PigGen Canada 60 k SNP chip Illumina GeneSeek2007
- 4. Partners and Funding Funding Industry Partners NIFA Iowa State University Nick Boddicker Andrew Hess Emily Waide, Jenelle Dunkelberger Eric Fritz-Waters James Koltes, Martine Schroyen Nick Serao Jim Reecy Chris Tuggle Susan Carpenter Kansas State University Bob Rowland PRRS group Ben Trible, Megan Niederwerder Maureen Kerrigan USDA-ARS Joan Lunney group, Igseo Choi, Sam Abrams University of Alberta Graham Plastow group Univ. Saskatchewan John Harding group Roslin Institute Steve Bishop Andrea Doeschl-Wilson Zeenath Islam Graham Lough Univ. Minnesota Monserat Torremorrell, Bob Morrison Scientific Collaborators
- 5. Integrated International Interdisciplinary Projects Nursery Pig PRRS infection PHGC, Genome Canada Nursery Pig PCV2 infection Ciobanu, Nebraska Gilt Acclimation Field challenges PigGen Canada, CSHB Pregnant Gilt PRRS infection Harding, Saskatchewan S A M P L E S + D A T A Project databases 60 SNP GWAS Transcriptomics Proteomics Genome Wide Associations Genomic Prediction Models of PRRS infection dynamics Improved vaccine targets Applications Kinomics In Vitro Assays Nursery Pig PRRS-PCV2 co-infection USDA-NIFA Growing Pig Field challenges USDA-NIFA
- 6. Nursery Pig Challenge Model Weight Serum Slaughter Ear for DNA -7 0 7 11 14 21 35 424 28 Serum Antibiotics Acclimation Birth Weight Serum Inoculation Weight Serum Weight Serum Weight Serum Weight Serum Weight Serum Serum Serum Day post infection R.R.R. Rowland et al., Kansas State University Groups of ~200 commercial crossbred pigs infected with PRRS virus isolate NVSL97-7985 between 18 and 28 d of age
- 7. Trial Number n Breed PRRSv Isolate 1-3 530 LW x LR NVSL 4 195 Duroc x LW/LR 5 184 Duroc x LR/LW 6 123 LR x LR 7 194 Pietran x LW/LR 8 188 Duroc x LW/LR 15 184 LR x LW 10 176 LR x LW KS06 11 176 LW x LR 12 174 LR x LW 14 180 Duroc x LR/LW 2304 challenged pigs with deep phenotypes and genotypes PHGC PRRS trials Generation 1 & 2
- 8. 0 1 2 3 4 5 6 0 5 10 15 20 25 30 Week Weight(kg) Host Response Phenotypes Body weight 0 4 7 11 14 21 28 35 42 0 2 4 6 8 Days Post Infection Viremia(Log) Log(viremia) Rebound Viral Load Area Under Curve h2 = 0.41h2 = 0.29 0 5 10 15 20 25 8090100110120 Weight Gain (kg) ViralLoad(Log) rp = -0.25 rg = -0.47
- 9. VIRAL LOAD WEIGHT GAIN Major QTL for host response to PRRS on SSC 4Boddicker et al. 2012, 2014a,b; Hess et al. 2015
- 10. 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 40 Log10(Viremia) Days Post Infection Viremia NVSL AA Viremia NVSL AB 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 40 Log10(Viremia) Days Post Infection Viremia KS06 AA Viremia KS06 AB Hess et al. 2015 Major QTL for host response to PRRS on SSC 4
- 11. 0 5 10 15 20 25 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 40 Weight(kg) Log10(Viremia) Days Post Infection Viremia NVSL AA Viremia NVSL AB Weight NVSL AA Weight NVSL AB 0 5 10 15 20 25 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 40 Weight(kg) Log10(Viremia) Days Post Infection Viremia KS06 AA Viremia KS06 AB Weight KS06 AA Weight KS06 AB Hess et al. 2015 Major QTL for host response to PRRS on SSC 4
- 12. AA pigs Truncated GBP5 protein PI3K-Akt pathway not turned off See Poster by Martine Schroyen et al. A Hypothesis
- 13. Genomic regions do not overlap between isolates but GO-term pathways do KS06 NVSL WUR = 21 WUR = 7WUR = 7 WUR = 21 See Poster by Emily Waide et al. Identification of other host response QTL Viral Load rg(NVSL, KS06) = 0.86 (+0.19)
- 14. KS06 NVSL WUR = 16WUR = 16 Weight Gain rg(NVSL, KS06) = 0.86 (+0.27) See Poster by Emily Waide et al. Identification of other host response QTL
- 15. Estimate SNP effectsSNP Genotypes Phenotypes SNP Genotypes Genomic Prediction Training population Validation population Phenotypes Genomic Prediction Prediction using high-density SNPs
- 16. KS06 NVSL Can we use Genomic Prediction across isolates? NVSL Trials KS06 Trials 17 r/h Waide et al. 2015
- 17. Total PRRS Antibody Response at day 42 MHC Class I MHC Class II Two SNPs in MHC explain 15 and 30% of genetic variance No association with WUR SNP on SSC4 h2 = 13% (Hess, Trible et al.)
- 18. 0.4 0.6 0.8 1 1.2 NVSL KS06 S:Pratio Cleared Rebound Persistent a b a a b a Relationship of PRRS Ab at 42 dpi with serum viremia profiles Hess et al. 2014
- 19. -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 5 10 15 20 25 30 35 40 Days Post Infection NVSL Viremia -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 5 10 15 20 25 30 35 40 Days Post Infection KS06 Viremia -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 5 10 15 20 25 30 35 40 Days Post Infection NVSL Weight Gain -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 5 10 15 20 25 30 35 40 Days Post Infection KS06 Weight Gain Phenotypic corr. Genetic correlation Correlations of S:P at 42 dpi with viremia and weight gain (Andrew Hess)
- 20. See Poster by Andrew Hess 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 NVSL KS06 Log10(TonsilViremia) Cleared Rebound Persistent n=203 n=71 n=206 n=94 n=13 n=195 a a,b b a a a Association of Tonsil viremia with serum viremial profile
- 21. Jack Dekkers, Chris Tuggle Jim Reecy, Ken Stalder Robert Rowland Yongming Sang Joan Lunney Montse Torremorell Robert Morrison Steve Bishop, David Hume Andrea Doeschl-Wilson Translational Genomics USDA-NIFA grant # 2013-68004-20362 3rd Generation PRRS Challenge Studies Co-infection and Field Studies
- 22. Genetically Improving Resistance of Pigs to PRRS Virus Infections Translational Genomics USDA-NIFA grant # 2013-68004-20362 Research Obj. 1 Obj. 2 Evaluate & validate gene c effects with co-infec on, PRRS vaccina on, and under fie l d condi ons Does the SSC4 QTL (+other QTL) • provide protec on under more realis c condi ons? • improve response to vaccina on? • Vaccine-ready pigs? Outreach Obj. 3 Education Obj. 4 Educate industry stakeholders and next generation of industry leaders
- 23. Arrive Vacci- nate Days post vaccination -1 0 4 7 11 14 21 28 32 35 39 42 49 56 63 70 Days post infection 0 4 7 11 14 21 28 35 42 Infect Vaccination response Vaccination + infection response Early response Later response Body weight and Blood samples 80k SNP genotypes Obj.1 Vaccination & Co-infection Trials Experimental design 4 groups of 200 commercial crossbred piglets (18-21 d) 50 AA Vaccinated (PRRS MLV) 50 AB 50 AA Non-vaccinated 50 AB WUR WUR Co-infection with PRRSv and PCV2 PCVAD
- 24. Serum Days post 0 7 14 21 28 42 60 90 Market arrival FMIA and SNA Serum Serum Serum Serum Weight Weight Weight Weight BF,LEA Body weight Pen oral fluids for disease surveillance purposes on all days indicated SNA on all oral fluid samples 29 Obj.2 Field Challenge Trials Experimental design 6 groups of 200 commercial crossbred piglets (18-21 d) 100 AA 100 AB WUR WUR Moved into health-challenged barns SNP genotypes
- 25. Pre PRRS VL PRRS VL Pre ADG Post ADG PCV2b VL ___ = AA Non Vx ___ = AB Non Vx ___ = AA Vx ___ = AB Vx Co-infection PRRS viremia Co-infectionCo-infection Body weight PCV2b viremia 50 AA Vaccinated (PRRS MLV) 50 AB 50 AA Non-vaccinated 50 AB WUR WUR Results 2 co-infection Trials VaccinationVaccination
- 26. See Poster by Jenelle Dunkelberger et al. Pre PRRS VL PRRS VL Post ADG Co-infection PRRS viremia Co-infectio PCV2b VL Vaccinatio Pre ADG Co-infection Vaccination 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Non-Vx Vx ADG(Kg/day) WUR Effect AA AB ** *** 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Non-Vx Vx ADG(Kg/day) WUR Effect *** 40 50 60 70 80 90 100 Non-Vx Vx ViralLoad WUR Effect ** 40 50 60 70 80 90 100 Non-Vx Vx ViralLoad WUR Effect ** *** * 70 80 90 100 110 120 130 140 150 160 Non-Vx Vx ViralLoad WUR Effect ** ** ___ = AA Non Vx ___ = AB Non Vx ___ = AA Vx ___ = AB Vx 50 AA Vaccinated (PRRS MLV) 50 AB 50 AA Non-vaccinated 50 AB WUR WUR Results 2 co-infection Trials
- 27. Pre PRRS VL PRRS VL Post ADG PCV2b VL Co-infection Co-infection Results 2 co-infection Trials Vaccination Pre ADG Co-infection Vaccination h2 rg Pre- NonVx Pre- Vx Post- NonVx Post- Vx Pre- NonVx 0.40 (0.20) 0.60 (0.64) 0.22 (0.36) -0.32 (0.39) Pre- Vx 0.16 (0.18) -0.07 (0.60) -0.47 (0.62) Post- NonVx 0.45 (0.20) 0.77 (0.36) Post- Vx 0.53 (0.22) h2 rg Pre- Vx Post- NonVx Post- Vx Pre- Vx 0.11 (0.20) 0.93 (1.06) 0.41 (0.97) Post- NonVx 0.54 (0.20) 0.21 (0.48) Post- Vx 0.18 (0.16) h2 rg Post- NonVx Post- Vx Post- NonVx 0.18 (0.15) 0.92 (0.50) Post- Vx 0.15 (0.15) r = 0.27 30 40 50 60 70 80 90 100 60 80 100 120 Post-… Pre-Infection Post- vs. Pre-PRRS VL Dunkelberger (2015)
- 28. 33 8,74 16,86 2,28 4,139 (WUR) 7,24 (MHC) 4,136 7,40 12,4 50 AA Vaccinated (PRRS MLV) 50 AB 50 AA Non-vaccinated 50 AB WUR WUR Genome Wide Association Studies See poster Jenelle Dunkelberger
- 29. Conclusions • Vaccination-co-infection design is good model for PCVAD • PRRSV vaccination amplification of PCVAD reduced growth • SSC4 QTL had a larger effect on PRRS viremia with primary exposure to PRRS innate immune function. • SSC4 QTL affected PCV2b viremia but only after PRRS vaccination • PRRS and PCV2b viremia controlled by different genomic regions with versus without vaccination. opportunities to select vaccine-ready pigs?
- 30. May 23-27, 2016, Iowa State University Dr. Andrea Doeschl-Wilson, Univ. Edinburgh 2014 course Disease Genetics: From Concepts to Utilization Prof. Steve Bishop, Univ. Edinburgh Disease Genetics Short Course Mathematical modelling of Infection Dynamics
- 31. Partners and Funding Funding Industry Partners NIFA Iowa State University Nick Boddicker Andrew Hess Emily Waide, Jenelle Dunkelberger Eric Fritz-Waters James Koltes, Martine Schroyen Nick Serao Jim Reecy Chris Tuggle Susan Carpenter Kansas State University Bob Rowland PRRS group Ben Trible, Megan Niederwerder Maureen Kerrigan USDA-ARS Joan Lunney group, Igseo Choi, Sam Abrams University of Alberta Graham Plastow group Univ. Saskatchewan John Harding group Roslin Institute Steve Bishop Andrea Doeschl-Wilson Zeenath Islam Graham Lough Univ. Minnesota Monserat Torremorrell, Bob Morrison Scientific Collaborators
Editor's Notes
- Results Within this QTL, guanylate binding protein 5 (GBP5) was differentially expressed (DE) (p < 0.05) in blood from AA versus AB rs80800372 genotyped pigs at 7,11, and 14 days post PRRSV infection. All variants within the GBP5 transcript in LD with rs80800372 exhibited allele specific expression (ASE) in AB individuals (p < 0.0001). A transcript re-assembly revealed three alternatively spliced transcripts for GBP5. An intronic SNP in GBP5, rs340943904, introduces a splice acceptor site that inserts five nucleotides into the transcript. Individuals homozygous for the unfavorable AA genotype predominantly produced this transcript, with a shifted reading frame and early stop codon that truncates the 88 C-terminal amino acids of the protein. RNA-seq analysis confirmed this SNP was associated with differential splicing by QTL genotype (p < 0.0001) and this was validated by quantitative capillary electrophoresis (p < 0.0001). The wild-type transcript was expressed at a higher level in AB versus AA individuals, whereas the five-nucleotide insertion transcript was the dominant form in AA individuals. Splicing and ASE results are consistent with the observed dominant nature of the favorable QTL allele. The rs340943904 SNP was also 100 % concordant with rs80800372 in a validation population that possessed an alternate form of the favorable B QTL haplotype. Conclusions GBP5 is known to play a role in inflammasome assembly during immune response. However, the role of GBP5 host genetic variation in viral immunity is novel. These findings demonstrate that rs340943904 is a strong candidate causal mutation for the SSC4 QTL that controls variation in host response to PRRSV.
- AB animals the full length GBP5 protein contains a CAAX box to anchor to the cell membrane, where it can bind to phosphoinositide 3 kinase (PI3K). The PI3K- Akt signal transduction pathway is involved in PRRSV entry and replication. By binding to PI3K, GBP5 inhibits its activation (Figure 4a). the system ‘thinks’ there is not enough PI3K and keeps the G-coupled protein receptor active as there is no feedback that it should be switched off, which leads to normal expression levels at 0 dpi. AA animals the truncated GBP5 protein does not bind to PI3K, hence it does not inhibit PI3K. Therefore, the virus can use the PI3K-Akt signal transduction pathway to enter and replicate in the host cell (Figure 4b). the system ‘thinks’ there is enough PI3K: feedback to switch the G-coupled protein receptor off, which leads to lower expression levels at 0 dpi.
- KS06 WG P 0.295 NVSL WG –log10(P) = 16
- KS06 WG P 0.295 NVSL WG –log10(P) = 16